This application claims priority from German Application No. 100 14 546.9, filed on Mar. 23, 2000, the subject matter of which is hereby incorporated herein by reference.
1. Field of the Invention
The invention provides nucleotide sequences encoding the dapC gene and a process for the fermentative production of L-lysine, using coryneform bacteria in which the dapC gene (N-succinylaminoketopimelate transaminase gene) is enhanced, in particular over-expressed.
2. Background Information
Amino acids, in particular L-lysine, are used in human medicine and in the pharmaceuticals industry, but in particular in animal nutrition.
It is known that amino acids are produced by fermentation of strains of coryneform bacteria, in particular Corynebacterium glutamicum. Due to their great significance, efforts are constantly being made to improve the production process. Improvements to the process may relate to measures concerning fermentation technology, for example stirring and oxygen supply, or to the composition of the nutrient media, such as for example sugar concentration during fermentation, or to working up of the product by, for example, ion exchange chromatography, or to the intrinsic performance characteristics of the microorganism itself.
The performance characteristics of these microorganisms are improved using methods of mutagenesis, selection and mutant selection. In this manner, strains are obtained which are resistant to antimetabolites, such as for example the lysine analogue S-(2-aminoethyl)cysteine, or are auxotrophic for regulatorily significant metabolites and produce L-amino acids, such as for example L-lysine.
For some years, methods of recombinant DNA technology have likewise been used to improve strains of Corynebacterium which produce amino acids by amplifying individual amino acid biosynthesis genes and investigating the effect on amino acid production. Review articles on this subject may be found inter alia in Kinoshita (xe2x80x9cGlutamic Acid Bacteriaxe2x80x9d, in: Biology of Industrial Microorganisms, Demain and Solomon (Eds.), Benjamin Cummings, London, UK, 1985, 115-142), Hilliger (BioTec 2, 40-44 (1991)), Eggeling (Amino Acids 6:261-272 (1994)), Jetten and Sinskey (Critical Reviews in Biotechnology 15, 73-103 (1995)) and Sahm et al. (Annuals of the New York Academy of Science 782, 25-39 (1996)).
It is an object of the invention to provide novel methods for the improved fermentative production of L-lysine.
L-lysine is used in human medicine, in the pharmaceuticals industry and in particular in animal nutrition. There is accordingly general interest in providing novel improved processes for the production of L-lysine.
Any subsequent mention of L-lysine or lysine should be taken to mean not only the base, but also salts, such as for example lysine monohydrochloride or lysine sulfate.
The invention provides an isolated polynucleotide from coryneform bacteria containing at least one polynucleotide sequence selected from the group
a) polynucleotide which is at least 70% identical to a polynucleotide which encodes a polypeptide containing the amino acid sequence of SEQ ID no. 2,
b) polynucleotide which encodes a polypeptide which contains an amino acid sequence which is at least 70% identical to the amino acid sequence of SEQ ID no. 2,
c) polynucleotide which is complementary to the polynucleotides of a) or b), or
d) polynucleotide containing at least 15 successive nucleotides of the polynucleotide sequences of a), b) or c).
The invention also provides the polynucleotide according to claim 1, wherein it preferably comprises replicable DNA containing:
(i) the nucleotide sequence shown in SEQ ID no. 1, or
(ii) at least one sequence which matches the sequence (i) within the degeneration range of the genetic code, or
(iii) at least one sequence which hybridizes with the complementary sequence to sequence (i) or (ii) and optionally
(iv) functionally neutral sense mutations in (i).
The invention also provides
a polynucleotide according to claim 4, containing the nucleotide sequence as shown in SEQ ID no. 1,
a polynucleotide which encodes a polypeptide which contains the amino acid sequence as shown in SEQ ID no. 2,
a vector containing the polynucleotide according to claim 1, in particular a shuttle vector or the plasmid vector pXT-dapCexp, which is shown in FIG. 2 and is deposited under number DSM 13254 in DSM 5715.
and coryneform bacteria acting as host cell which contain the vector.
The invention also provides polynucleotides which substantially consist of a polynucleotide sequence, which are obtainable by screening by means of hybridization of a suitable gene library, which contains the complete gene having the polynucleotide sequence according to SEQ ID no. 1, with a probe which contains the sequence of the stated polynucleotide according to SEQ ID no. 1, or a fragment thereof, and isolation of the stated DNA sequence.
Polynucleotide sequences according to the invention are suitable as hybridization probes for RNA, cDNA and DNA in order to isolate full length cDNA which encode N-succinylaminoketopimelate transaminase and to isolate such cDNA or genes, the sequence of which exhibits a high level of similarity with that of the N-succinylaminoketopimelate transaminase gene.
Polynucleotide sequences according to the invention are furthermore suitable as primers for the production of DNA of genes which encode N-succinylaminoketopimelate transaminase by the polymerase chain reaction (PCR).
Such oligonucleotides acting as probes or primers contain at least 30, preferably at least 20, very particularly preferably at least 15 successive nucleotides. oligonucleotides having a length of at least 40 or 50 nucleotides are also suitable.
xe2x80x9cIsolatedxe2x80x9d means separated from its natural environment.
xe2x80x9cPolynucleotidexe2x80x9d generally relates to polyribonucleotides and polydeoxyribonucleotides, wherein the RNA or DNA may be unmodified or modified.
xe2x80x9cPolypeptidesxe2x80x9d are taken to mean peptides or proteins which contain two or more amino acids connected by peptide bonds.
The polypeptides according to the invention include a polypeptide according to SEQ ID no. 2, in particular those having the biological activity of N-succinylaminoketopimelate transaminase and also those which are at least 70%, preferably at least 80%, identical to the polypeptide according to SEQ ID no. 2 and in particular are at least 90% to 95% identical to the polypeptide according to SEQ ID no. 2 and exhibit the stated activity.
The invention furthermore relates to a process for the fermentative production of amino acids, in particular L-lysine, using coryneform bacteria, which in particular already produce an amino acid and in which the nucleotide sequences which encode the dapC gene are enhanced, in particular over-expressed.
In this connection, the term xe2x80x9cenhancementxe2x80x9d describes the increase in the intracellular activity of one or more enzymes in a microorganism, which enzymes are encoded by the corresponding DNA, for example by increasing the copy number of the gene or genes, by using a strong promoter or a gene or allele which encodes a corresponding enzyme having elevated activity and optionally by combining these measures.
The microorganisms, provided by the present invention, may produce L-amino acids, in particular L-lysine, from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol. The microorganisms may comprise representatives of the coryneform bacteria in particular of the genus Corynebacterium. Within the genus Corynebacterium, the species Corynebacterium glutamicum may in particular be mentioned, which is known in specialist circles for its ability to produce L-amino acids.
Suitable strains of the genus Corynebacterium, in particular of the species Corynebacterium glutamicum, are for example the known wild type strains
Corynebacterium glutamicum ATCC13032
Corynebacterium acetoglutamicum ATCC15806
Corynebacterium acetoacidophilum ATCC13870
Corynebacterium thermoaminogenes FERM BP-1539
Corynebacterium melassecola ATCC17965
Brevibacterium flavum ATCC14067
Brevibacterium lactofermentum ATCC13869 and
Brevibacterium divaricatum ATCC14020
and L-lysine producing mutants or strains produced therefrom, such as for example
Corynebacterium glutamicum FERM-P 1709
Brevibacterium flavum FERM-P 1708
Brevibacterium lactofermentum FERM-P 1712
Corynebacterium glutamicum FERM-P 6463
Corynebacterium glutamicum FERM-P 6464
Corynebacterium glutamicum DSM5715
Corynebacterium glutamicum DSM12866 and
Corynebacterium glutamicum DM58-1.
The inventors succeeded in isolating the novel dapC gene, which encodes the enzyme N-succinylaminoketopimelate transaminase (EC 2.6.1.17), from C. glutamicum. 
The dapC gene, and also other genes from C. glutamicum, are isolated by initially constructing a gene library of this microorganism in E. coli. The construction of gene libraries is described in generally known textbooks and manuals. Examples which may be mentioned are the textbook by Winnacker, Gene und Klone, Eine Einfxc3xchrung in die Gentechnologie (Verlag Chemie, Weinheim, Germany, 1990) or the manual by Sambrook et al., Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989). One very well known gene library is that of E. coli K-12 strain W3110, which was constructed by Kohara et al. (Cell 50, 495-508 (1987)) in xcex-vectors. Bathe et al. (Molecular and General Genetics, 252:255-265, 1996) describe a gene library of C. glutamicum ATCC13032, which was constructed using the cosmid vector SuperCos I (Wahl et al., 1987, Proceedings of the National Academy of Sciences USA, 84:2160-2164) in E. coli K-12 strain NM554 (Raleigh et al., 1988, Nucleic Acids Research 16:1563-1575). Bxc3x6rmann et al. (Molecular Microbiology 6(3), 317-326, 1992)) also describe a gene library of C. glutamicum ATCC 13032, using cosmid pHC79 (Hohn and Collins, Gene 11, 291-298 (1980)). A gene library of C. glutamicum in E. coli may also be produced using plasmids such as pBR322 (Bolivar, Life Sciences, 25, 807-818 (1979)) or pUC9 (Vieira et al., 1982, Gene, 19:259-268). Suitable hosts are in particular those E. coli strains with restriction and recombination defects. One example of such a strain is the strain DH5xcex1mcr, which has been described by Grant et al. (Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649). The long DNA fragments cloned with the assistance of cosmids may then in turn be sub-cloned in usual vectors suitable for sequencing and then be sequenced, as described, for example, in Sanger et al. (Proceedings of the National Academy of Sciences of the United States of America, 74:5463-5467, 1977).
The novel DNA sequence from C. glutamicum which encodes the dapC gene and, as SEQ ID no. 1, is provided by the present invention, was obtained in this manner. The amino acid sequence of the corresponding protein was furthermore deduced from the above DNA sequence using the methods described above. SEQ ID no. 2 shows the resultant amino acid sequence of the product of the dapC gene.
Coding DNA sequences arising from SEQ ID no. 1 due to the degeneracy of the genetic code are also provided by the invention. DNA sequences which hybridize with SEQ ID no. 1 or parts of SEQ ID no. 1 are also provided by the invention. Conservative substitutions of amino acids in proteins, for example the substitution of glycine for alanine or of aspartic acid for glutamic acid, are known in specialist circles as xe2x80x9csense mutationsxe2x80x9d, which result in no fundamental change in activity of the protein, i.e. they are functionally neutral. It is furthermore known that changes to the N and/or C terminus of a protein do not substantially impair or may even stabilize the function thereof. The person skilled in the art will find information in this connection inter alia in Ben-Bassat et al. (Journal of Bacteriology 169:751-757 (1987)), in O""Regan et al. (Gene 77:237-251 (1989)), in Sahin-Toth et al. (Protein Sciences 3:240-247 (1994)), in Hochuli et al. (Bio/Technology 6:1321-1325 (1988)) and in known textbooks of genetics and molecular biology. Amino acid sequences arising in a corresponding manner from SEQ ID no. 2 are also provided by the invention.
Similarly, DNA sequences which hybridize with SEQ ID no. 1 or portions of SEQ ID no. 1 are also provided by the invention. Finally, DNA sequences produced by the polymerase chain reaction (PCR) using primers obtained from SEQ ID no. 1 are also provided by the invention. Such oligonucleotides typically have a length of at least 15 nucleotides.
The person skilled in the art may find instructions for identifying DNA sequences by means of hybridization inter alia in the manual xe2x80x9cThe DIG System Users Guide for Filter Hybridizationxe2x80x9d from Roche Diagnostics GmbH (Mannheim, Germany, 1993) and in Liebl et al. (International Journal of Systematic Bacteriology (1991) 41: 255-260). The person skilled in the art may find instructions for amplifying DNA sequences using the polymerase chain reaction (PCR) inter alia in the manual by Gait, Oligonucleotide Synthesis: A Practical Approach (IRL Press, Oxford, UK, 1984) and in Newton and Graham, PCR (Spektrum Akademischer Verlag, Heidelberg, Germany, 1994).
It has been found that coryneform bacteria produce L-lysine in an improved manner once the dapC gene has been over-expressed.
Over-expression may be achieved by increasing the copy number of the corresponding genes or by mutating the promoter and regulation region or the ribosome-binding site located upstream from the structural gene. Expression cassettes incorporated upstream from the structural gene act in the same manner. It is additionally possible to increase expression during fermentative L-lysine production by means of inducible promoters. Expression is also improved by measures to extend the lifetime of the mRNA. Enzyme activity is moreover enhanced by preventing degradation of the enzyme protein. The genes or gene constructs may either be present in plasmids in a variable copy number or be integrated in the chromosome and amplified. Alternatively, over-expression of the genes concerned may also be achieved by modifying the composition of the media and culture conditions.
The person skilled in the art will find guidance in this connection inter alia in Martin et al. (Bio/Technology 3, 137-146 (1987)), in Guerrero et al. (Gene 138, 35-41 (1994)), Tsuchiya and Morinaga (Bio/Technology 6, 428-430 (1988)), in Eikmanns et al. (Gene 102, 93-98 (1991)), in EP 0 472 869, in U.S. Pat. No. 4,601,893, in Schwarzer and Pxc3xchler (Bio/Technology 9, 84-87 (1991), in Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)), in LaBarre et al. (Journal of Bacteriology 175, 1001-1007 (1993)), in WO 96/15246, in Malumbres et al. (Gene 134, 15-24 (1993)), in JP-A-10-229891, in Jensen and Hammer (Biotechnology and Bioengineering 58, 191-195 (1998)), in Makrides (Microbiological Reviews 60:512-538 (1996)) and in known textbooks of genetics and molecular biology.
By way of example, the dapC gene according to the invention was over-expressed with the assistance of plasmids.
Suitable plasmids are those which are replicated in coryneform bacteria. Numerous known plasmid vectors, such as for example pZ1 (Menkel et al., Applied and Environmental Microbiology (1989) 64: 549-554), pEKEx1 (Eikmanns et al., Gene 102:93-98 (1991)) or pHS2-1 (Sonnen et al., Gene 107:69-74 (1991)) are based on the cryptic plasmids pHM1519, pEL1 or pGA1. Other plasmid vectors, such as for example those based on pCG4 (U.S. Pat. No. 4,489,160), or pNG2 (Serwold-Davis et al., FEMS Microbiology Letters 66, 119-124 (1990)), or pAG1 (U.S. Pat. No. 5,158,891) may be used in the same manner.
One example of a plasmid by means of which the dapC gene may be over-expressed is the E. coli-C. glutamicum shuttle vector pXT-dapCexp. The vector contains the replication region rep of plasmid pGA1, including the replication effector per (U.S. Pat. No. 5,175,108; Nesvera et al., Journal of Bacteriology 179, 1525-1532 (1997)), the tetA(Z) gene, which imparts tetracycline resistance, of plasmid pAG1 (U.S. Pat. No. 5,158,891; GenBank entry at the National Center for Biotechnology Information (NCBI, Bethesda, Md., USA) with the accession number AF121000), together with the replication origin, the trc promoter, the termination regions T1 and T2 and the lacIq gene (repressor of the lac operon of E. coli) of plasmid pTRC99A (Amann et al. (1988), Gene 69: 301-315).
The shuttle vector pXT-dapCexp is shown in FIG. 2.
Further suitable plasmid vectors are those with the assistance of which gene amplification may be performed by integration into the chromosome, as has for example been described by Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)) for the duplication or amplification of the hom-thrB operon. In this method, the complete gene is cloned into a plasmid vector which can replicate in a host (typically E. coli), but not in C. glutamicum. Vectors which may be considered are, for example, pSUP301 (Simon et al., Bio/Technology 1, 784-791 (1983)), pK18mob or pK19mob (Schxc3xa4fer et al., Gene 145, 69-73 (1994)), pGEM-T (Promega corporation, Madison, Wis., USA), pCR2.1-TOPO (Shuman (1994). Journal of Biological Chemistry 269:32678-84; U.S. Pat. No. 5,487,993), pCR(copyright)Blunt (Invitrogen, Groningen, Netherlands; Bernard et al., Journal of Molecular Biology, 234: 534-541 (1993)) or pEM1 (Schrumpf et al, 1991, Journal of Bacteriology 173:4510-4516). The plasmid vector which contains the gene to be amplified is then transferred into the desired strain of C. glutamicum by conjugation or transformation. The conjugation method is described, for example, in Schxc3xa4fer et al. (Applied and Environmental Microbiology 60, 756-759 (1994)). Transformation methods are described, for example, in Thierbach et al. (Applied Microbiology and Biotechnology 29, 356-362 (1988)), Dunican and Shivnan (Bio/Technology 7, 1067-1070 (1989)) and Tauch et al. (FEMS Microbiological Letters 123, 343-347 (1994)). After homologous recombination by means of xe2x80x9ccrossing overxe2x80x9d, the resultant strain contains at least two copies of the gene in question.
It has furthermore been found that, by replacing the amino acid L-proline in position 209 of the enzyme protein (c.f. SEQ ID no. 2) with another proteinogenic amino acid, in particular L-leucine (c.f. SEQ ID no. 4), with the exception of L-proline, enhancement occurs and coryneform bacteria bearing the corresponding amino acid replacement produce L-lysine in an improved manner. The replacement of L-proline with L-leucine in position 209 may preferably be achieved by replacing the nucleobase cytosine in position 716 with thymine as shown in SEQ ID no. 3.
Mutagenesis may be performed by conventional mutagenesis methods using mutagens such as for example N-methyl-Nxe2x80x2-nitro-N-nitrosoguanidine or ultraviolet light. Mutagenesis may also be performed by using in vitro methods such as for example treatment with hydroxylamine (Molecular and General Genetics 145, 101 pp (1978)) or mutagenic oligonucleotides (T. A. Brown: Gentechnologie fur Einsteiger, Spektrum Akademischer Verlag, Heidelberg, 1993) or the polymerase chain reaction (PCR), as is described in the manual by Newton and Graham (PCR, Spektrum Akademischer Verlag, Heidelberg, 1994).
The invention accordingly also provides DNA originating from coryneform bacteria which encodes N-succinylaminoketopimelate transaminase, in which the amino acid sequence shown in SEQ ID no. 2 in position 209 is replaced with another amino acid, with the exception of L-proline. The invention also relates to coryneform bacteria which contain DNA in which the amino acid L-proline in position 209 of the enzyme protein (c.f. SEQ ID no. 2) is replaced with L-leucine (c.f. SEQ ID no. 4).
The invention furthermore provides coryneform bacteria which contain DNA in which the replacement of L-proline with L-leucine in position 209 proceeds by the replacement of the nucleobase cytosine in position 716 with thymine, as shown in SEQ ID no. 3.
It may additionally be advantageous for the production of L-lysine to amplify or over-express not only the dapC gene, but also one or more enzymes of the particular biosynthetic pathway, of glycolysis, of anaplerotic metabolism, or of amino acid export.
For the production of L-lysine, for example, it is thus possible in addition to the dapC gene simultaneously to enhance, in particular over-express or amplify, one or more genes selected from the group
the lysC gene, which encodes a feed-back resistant aspartate kinase (Kalinowski et al. (1990), Molecular and General Genetics 224, 317-324),
the asd gene, which encodes aspartate semialdehyde dehydrogenase (EP-A 0 219 027; Kalinowski et al. (1991), Molecular Microbiology 5:1197-1204, and Kalinowski et al.(1991), Molecular and General Genetics 224: 317-324),
the dapA gene, which encodes dihydropicolinate synthase (EP-B 0 197 335),
the dapB gene, which encodes dihydrodipicolinate reductase (GenBank entry accession number X67737; Pisabarro et al. (1993), Journal of Bacteriology, 175(9): 2743-2749),
the dapD gene, which encodes tetrahydrodipicolinate succinylase (GenBank entry accession number AJ004934; Wehrmann et al. (1998), Journal of Bacteriology 180: 3159-3163),
the dapE gene, which encodes N-succinyldiaminopimelate desuccinylase (GenBank entry accession number X81379; Wehrmann et al. (1994), Microbiology 140: 3349-3356),
the dapF gene, which encodes diaminopimelate epimerase (DE: 199 43 587.1, DSM12968),
the lysA gene, which encodes diaminopimelate decarboxylase (GenBank entry accession number X07563; Yeh et al. (1988), Molecular and General Genetics 212: 112-119),
the ddh gene, which encodes diaminopimelate dehydrogenase (Ishino et al. (1988), Agricultural and Biological Chemistry 52(11): 2903-2909),
the lysE gene, which encodes lysine export (DE-A-195 48 222),
the pyc gene, which encodes pyruvate carboxylase (Eikmanns (1992), Journal of Bacteriology 174: 6076-6086),
the mqo gene, which encodes malate:quinone oxidoreductase (Molenaar et al. (1998), European Journal of Biochemistry 254: 395-403),
the zwa1 gene (DE: 19959328.0, DSM 13115),
the gdh gene, which encodes glutamate dehydrogenase (Bxc3x6rmann et al. (1992), Molecular Microbiology 6, 317-326).
It is preferred simultaneously to enhance one or more genes selected from the group dapD, dapE and dapF.
It may furthermore be advantageous for the production of L-lysine, in addition to enhancing the dapC gene, optionally in combination with one or more genes selected from the group dapD, dapE and dapF, simultaneously to attenuate
the pck gene, which encodes phosphoenolpyruvate carboxykinase (DE 199 50 409.1, DSM 13047) or
the pgi gene, which encodes glucose 6-phosphate isomerase (U.S. Ser. No. 09/396,478, DSM 12969), or
the poxB gene, which encodes pyruvate oxidase (DE: 19951975.7, DSM 13114), or
the zwa2 gene (DE: 19959327.2, DSM 13113), or
the sucC or sucD genes which encode succinyl CoA synthetase (DE: 19956686.0).
It may furthermore be advantageous for the production of L-lysine, in addition to enhancing the dapC gene, optionally in combination with one or more genes selected from the group dapD, dapE and dapF, to suppress unwanted secondary reactions (Nakayama: xe2x80x9cBreeding of Amino Acid Producing Micro-organismsxe2x80x9d, in: Over-production of Microbial Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK, 1982).
For the purposes of L-lysine production, the microorganisms produced according to the invention may be cultured continuously or discontinuously using the batch process or the fed batch process or repeated fed batch process. A summary of known culture methods is given in the textbook by Chmiel (Bioprozesstechnik 1. Einfuhrung in die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktoren und periphere Einrichtungen (Vieweg Verlag, Braunschweig/Wiesbaden, 1994)).
The culture medium to be used must adequately satisfy the requirements of the particular strains. Culture media for various microorganisms are described in xe2x80x9cManual of Methods for General Bacteriologyxe2x80x9d from the American Society for Bacteriology (Washington D.C., USA, 1981).
Carbon sources which may be used are sugars and carbohydrates, such as glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose for example, oils and fats, such as soya oil, sunflower oil, peanut oil and coconut oil for example, fatty acids, such as palmitic acid, stearic acid and linoleic acid for example, alcohols, such as glycerol and ethanol for example, and organic acids, such as acetic acid for example. These substances may be used individually or as a mixture.
Nitrogen sources which may be used comprise organic compounds containing nitrogen, such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soya flour and urea or inorganic compounds, such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. The nitrogen sources may be used individually or as a mixture.
Phosphorus sources which may be used are phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding salts containing sodium. The culture medium has additionally to contain salts of metals, such as magnesium sulfate or iron sulfate for example, which are necessary for growth. Finally, essential growth-promoting substances such as amino acids and vitamins may also be used in addition to the above-stated substances. Suitable precursors may furthermore be added to the culture medium. The stated feed substances may be added to the culture as a single batch or be fed appropriately during culturing.
Basic compounds, such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water, or acidic compounds, such as phosphoric acid or sulfuric acid, are used appropriately to control the pH of the culture Foaming may be controlled by using antifoaming agents such as fatty acid polyglycol esters for example. Plasmid stability may be maintained by the addition to the medium of suitable selectively acting substances, for example antibiotics. Oxygen or oxygen-containing gas mixtures, such as air for example, are introduced into the culture in order to maintain aerobic conditions. The temperature of the culture is normally from 20xc2x0 C. to 45xc2x0 C. and preferably from 25xc2x0 C. to 40xc2x0 C. The culture is continued until the maximum quantity of lysine has formed. This aim is normally achieved within 10 to 160 hours.
Analysis of L-lysine may be performed by anion exchange chromatography with subsequent ninhydrin derivation, as described in Spackman et al. (Analytical Chemistry, 30, (1958), 1190).
The following microorganism has been deposited with Deutsche Sammlung fxc3xcr Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany) in accordance with the Budapest Treaty:
Corynebacterium glutamicum strain DSM5715/pXT-dapCexp as DSM 13254.
The process according to the invention serves in the fermentative production of L-lysine.